Enzymatic production of peracids from carboxylic acid ester substrates using non-heme haloperoxidases

Abstract

A process is provided for producing peroxycarboxylic acids from carboxylic acid esters. More specifically, carboxylic acid esters are reacted in situ with an inorganic peroxide in the presence of a non-heme haloperoxidase having perhydrolysis activity to produce peroxycarboxylic acids.

1 . A process for producing peracids from carboxylic acid ester substrates comprising 
 a) providing a set of peracid reaction components, said components comprising: 
 1) a carboxylic acid ester substrate selected from the group consisting of: 
 i) esters having the structure  
                     
 wherein R 1 =C1 to C10 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 2 =C1 to C10 straight chain or branched chain alkyl group, (CH 2 CH 2 —O) n H or (CH 2 CH(CH 3 )—O) n H and n=1 to 10; and  
 ii) glycerides having the structure  
                     
 wherein R 1 =C1 to C10 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 3  and R 4  are individually H or R 1 C(O); and  
 
 2) a source of peroxygen; and  
 3) a non-heme haloperoxidase catalyst having a perhydrolysis activity; and  
   b) combining said reaction components under aqueous reaction conditions, wherein said conditions comprising a pH range of about 2 to about 7.5, whereby a peracid is produced at a concentration of at least 500 ppb within about 5 minutes to about 2 hours of combining the reaction components.    
     
     
         2 . The process of  claim 1  wherein the pH is less than about 6.5.  
     
     
         3 . The process of  claim 2  wherein the pH range is less than about 4.5.  
     
     
         4 . The process of claims  1 - 3  wherein the ester substrate is selected from the group consisting of methyl lactate, ethyl lactate, methyl glycolate, ethyl glycolate, methyl methoxyacetate, ethyl methoxyacetate, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, and mixtures thereof.  
     
     
         5 . The process of claims  1 - 3  wherein the glyceride is selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, glyceryl dipropionate, tripropionin, monobutyrin, glyceryl dibutyrate, tributyrin, and mixtures thereof.  
     
     
         6 . The process of  claim 4  or  claim 5  wherein the non-heme haloperoxidase is derived from an organism selected from the group consisting of  Pseudomonas putida  and  Agrobacterium tumefaciens.    
     
     
         7 . The process of  claim 6  wherein the non-heme haloperoxidase is derived from a microorganism selected from the group consisting of  Psuedomonas putida  5B (NRRL-1 8668) and  Agrobacterium tumefaciens  C58 (ATCC 33970).  
     
     
         8 . The process of  claim 7  wherein the non-heme haloperoxidase having perhydrolysis activity is encoded by an isolated nucleic acid molecule is selected from the group consisting of: 
 a) an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO:  22 , and SEQ ID NO: 28; and    b) an isolated nucleic acid molecule encoding a polypeptide having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 28.    
     
     
         9 . The process of  claim 4  wherein the peracid produced is selected from the group consisting of peracetic acid, perpropionic acid, perbutyric acid, perlactic acid, perglycolic acid, permethoxyacetic acid, per-β-hydroxybutyric acid, and mixtures thereof.  
     
     
         10 . The process of  claim 9  wherein the peracid produced is peracetic acid.  
     
     
         11 . A process to reduce a microbial population on a hard surface or inanimate object using an enzymatically produced aqueous peracid composition, said process comprising: 
 a) providing a set of peracid reaction components, said components comprising: 
 1. a substrate selected from the group consisting of: 
 i) esters having the structure  
                     
 wherein R 1 =C1 to C10 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 2 =C1 to C10 straight chain or branched chain alkyl group, (CH 2 CH 2 —O) n H or (CH 2 CH(CH 3 )—O) n H and n=1 to 10; and  
 ii) glycerides having the structure  
                     
 wherein R 1 =C1 to C10 straight chain or branched chain alkyl optionally substituted with an hydroxyl or a C1 to C4 alkoxy group and R 3  and R 4  are individually H or R 1 C(O); and  
 
 2) a source of peroxygen; and  
 3) a non-heme haloperoxidase catalyst having perhydrolysis activity;  
   b) providing a hard surface or an inanimate object having a concentration of microorganisms and/or viruses;    c) combining said reaction components under aqueous reaction conditions, wherein said conditions comprising a pH range of about 2 to about 7.5, whereby a peracid product is formed having a peracid concentration of at least 500 ppb within about 5 minutes to about 2 hours of combining the reaction components;    d) optionally diluting the said peracid product; and    e) contacting said hard surface or said inanimate object with the peracid produced in step c) or step d) within 48 hours of combining said reaction components whereby the concentration of said of microorganisms is reduced at least 3-log.    
     
     
         12 . The process of  claim 11  wherein the hard surface or the inanimate object is contacted with the peracid produced in step c) or step d) within about 30 minutes of combining said reaction components.  
     
     
         13 . The process of  claim 12  wherein the hard surface or the inanimate object is contacted with the peracid produced in step c) or step d) within about 10 minutes of combining said reaction components.  
     
     
         14 . The process according to  claims 11  to  13  wherein the concentration of said microorganisms is reduced at least 4-log.  
     
     
         15 . The process of  claim 11  wherein the pH range is less than about 6.5.  
     
     
         16 . The process of  claim 15  wherein the pH range is less than about 4.5.  
     
     
         17 . The process of  claim 11  wherein the ester substrate is selected from the group consisting of methyl lactate, ethyl lactate, ethyl acetate, methyl glycolate, ethyl glycolate, methyl methoxyacetate, ethyl methoxyacetate, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, and mixtures thereof.  
     
     
         18 . The process of  claim 17  wherein the ester substrate is selected from the group consisting of ethyl lactate, ethyl acetate, and mixtures thereof.  
     
     
         19 . The process of  claim 11  wherein the glyceride substrate is selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, glyceryl dipropionate, tripropionin, monobutyrin, glyceryl dibutyrate, tributyrin, and mixtures thereof.  
     
     
         20 . The process of  claim 19  wherein the glycercide substrate is selected from the group consisting of diacetin and triacetin.  
     
     
         21 . The process of  claim 11  wherein the non-heme haloperoxidase is derived from an organism selected from the group consisting of  Pseudomonas putida  and  Agrobacterium tumefaciens.    
     
     
         22 . The process of  claim 21  wherein the non-heme haloperoxidase is derived from a microorganism selected from the group consisting of  Psuedomonas putida  5B (NRLL-18668) and  Agrobacterium tumefaciens  C58 (ATCC 33970).  
     
     
         23 . The process of  claim 22  wherein the non-heme haloperoxidase having perhydrolysis activity is encoded by an isolated nucleic acid molecule is selected from the group consisting of: 
 a) an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO:  22 , and SEQ ID NO: 28; and    b) an isolated nucleic acid molecule encoding a polypeptide having at least 95% identity to an amino acid sequence selected form the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 28.    
     
     
         24 . The process of claims  23  wherein the peracid is produced at a concentration of at least 500 ppb within about 2 hours.  
     
     
         25 . The process of  claim 24  wherein the peracid is produced at a concentration of at least 1 ppm within about 2 hours.  
     
     
         26 . The process of  claim 25  wherein the peracid is produced at a concentration of at least 5 ppm within about 2 hours.  
     
     
         27 . The process of  claim 11  or 23 wherein the peracid produced is selected from the group consisting of peracetic acid, perpropionic acid, perbutyric acid, perlactic acid, perglycolic acid, permethoxyacetic acid, perβ-hydroxybutyric acid, and mixtures thereof.  
     
     
         28 . The process according to  claim 27  wherein the peracid produced is peracetic acid.  
     
     
         29 . An isolated nucleic acid molecule encoding a polypeptide having perhydrolytic activity wherein said polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 10.  
     
     
         30 . The isolated nucleic acid molecule of  claim 29  encoding a polypeptide having an amino acid sequence as represented by SEQ ID NO: 10.  
     
     
         31 . The process of  claim 11 , further comprising combining the peracid product of step (c), with one or more additional agents, including an antiseptic, disinfectant, virucide, biocide, antimicrobial agent, or mixtures thereof.  
     
     
         32 . The process of  claim 31 , wherein the additional agent is a virucide selected from the group consisting of alcohols, ethers, chloroform, formaldehyde, phenols, beta propiolactone, iodine, chlorine, mercury salts, hydroxylamine, ethylene oxide, ethylene glycol, quaternary ammonium compounds, enzymes, and detergents.  
     
     
         33 . The process of  claim 31 , wherein the additional agent is a biocide selected from the group consisting of chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone, acrolein, amines, chlorinated phenolics, copper salts, organo-sulphur compounds, and quaternary ammonium salts.